The ability to detect and quantify ligands and receptors (individually and collectively referred to as specific binding pair members) has become increasingly important. A variety of clinical conditions may be diagnosed and monitored by detecting the presence of and/or amount of a specific binding pair member in a sample. Examples of ligands which have a receptor includes compounds having either natural or synthetic activity. Such compounds may have low molecular weights (i.e., 125-2,000), such as small polypeptides, enzyme substrates, lipids and hormones, or may have higher molecular weights (i.e., &gt;5,000), such as proteins, nucleic acids and glycoproteins. More specifically, such higher molecular weight compounds may include immunoglobulins and further may include monoclonal antibodies.
As the importance of measuring the presence of a specific binding pair member in a sample has increased, a number of means have been developed to detect such members. One method involves the direct conjugation of a label to the specific binding pair member. This labelled material then is allowed to tag the other specific binding pair member in a sample. The usefulness of the labelled material, however, will depend upon the specificity of the specific binding pair member for the other member, and also will depend upon the non-specific binding of the labelled material. The greater the non-specific binding of the labelled material, the lesser the sensitivity of the measurement will be.
Of great interest today is the use of fluorochromes as a label particularly when directly conjugated to monoclonal antibodies. When a fluorochrome is coupled to a monoclonal antibody the material may be used to label a cell bearing a specific receptor. The labelled material once bound to the specific receptor then may be detected by one of several means including a fluorescence light microscope and flow cytometer. U.S. Pat. No. 4,520,111 describes the conjugation of specific binding pair members to phycobiliproteins, and particularly to phycoerythrin (PE). U.S. Pat. No. 4,520,111 also describes the use of such labelled materials to tag lymphocytes and to be detected using flow cytometry. U.S. Pat. No. 4,542,104 further describes the conjugation of one or more fluorochromes to each other which then may be bound to a member of a specific binding pair.
In all of these cases, however, and in examples of conjugates not specifically described, the labelled material will bind non specifically. Depending upon the amount of non-specific staining, therefore, background fluorescence may cover up or mask the fluorescence signal from the labelled material specifically bound to the receptor. The degree of background or non-specific binding will limit the usefulness of the labelled material.
Non-specific binding does not appear to be a function of the method by which labelled material is prepared. Non-specific binding appears to result from the methods used to prepare the specific binding pair member to be detected (or material containing the member) prior to combining the labelled material with the member to be detected.
For example, in the preparation of whole blood lymphocytes for subset analyses by flow cytometry, erythrocytes are removed in order to leave a population of peripheral blood leukocytes (PBL). One method is to lyse the erythrocytes with a lysing solution. Such lysing solutions must be sufficiently strong to lyse the erythrocytes but must not be so strong to lyse the PBL. One solution that achieves these results is described in U.S. Pat. No. 4,654,312, and comprises an aqueous solution of a short chain (1-4) aliphatic aldehyde, an alkali or alkaline earth salt of a weak acid and a polyhydric alcohol.
The method of using this lysing solution generally comprises taking a sample of peripheral blood from an individual, adding a labelled material (e.g., anti-CD14 (PE)) to an aliquot of blood for a period of time sufficient to allow binding to the CD14 receptor, adding the lysing solution for a period of time sufficient to lyse the erythrocytes, centrifuging and washing the resulting mixture, adding a diluent to the mixture and placing the mixture into a flow cytometer. Modification to this method may include the addition of multiple labelled materials to the aliquot wherein, for example, each member binds to a different receptor and each fluorochrome has a different peak emission spectra, and/or the addition of dyes which differentially bind to nucleic acids. U.S. Pat. No. 4,654,312 describes one such method.
The limitation of this method, however, is that some non-specific binding will occur. As a result, a new method and material to reduce the non-specific binding of labelled material is needed.